The gene under investigation in this study, MAL (myelin and lymphocyte protein), has been implicated in several malignancies including esophageal, ovarian and cervical cancers. The MAL protein is involved in apical transport in polarized-epithelial cells and therefore its disruption may lead to the loss of organized polarity, a characteristic of most solid malignancies. Aberrant methylation of gene promoters leading to transcriptional silencing is associated with the onset and progression of cancer. Preliminary data we have accumulated thus far demonstrates that the MAL promoter is frequently hypermethylated in breast cancer but unmethylated in normal breast epithelial cells. In this study we will be investigating the significance and consequences of this finding. Based on our preliminary findings, we hypothesize that MAL promoter hypermethylation blocks the binding of transcriptional regulatory proteins resulting in transcriptional silencing and the disruption of normal biological activities that contribute to breast cancer development and progression. Specific aim #1 will determine the nature and frequency of hypermethylation in the MAL promoter and whether these methylation events affect gene expression. The main experiments for this aim include bisulfite genomic sequencing, methylation-specific PCR (MSP) and RT-PCR expression analysis following treatment with the methylation inhibitor decitabine. Specific aim #2 will determine whether methylation silences gene expression by blocking the binding of gene regulatory proteins. We will use chromatin immunoprecipitation analysis to monitor transcription factor binding under varying conditions and luciferase gene expression analysis to observe changes in expression after protein binding. Specific aim #3 will determine the biological consequences of MAL expression such as changes in cell cycle and apoptosis, and whether methylation affects the biological activity. Specific aim #4 will assess MAL's use as a biomarker for detection based on the ability to detect MAL promoter methylation in serum from breast cancer patients. Our preliminary data indicate that hypermethylation of MAL is a common event in primary breast cancers and may be a useful marker for detection. Its role in cell polarity may define a new functional class of tumor suppressor genes and thus warrants further study with respect to the development and etiology of cancer.